In the series, Frasier shared a luxurious condominium apartment with his father, Martin (played by John Mahoney), and Martin's live-in physical therapist, Daphne Moon (played by Jane Leeves). Niles, a frequent visitor, soon became enamored of Daphne.

The match seemed an unlikely one since Niles was sophisticated, even snobbish--but still likable--while Daphne was a working-class woman with simpler tastes, although being British did give her a touch of the exotic. Many glbtq viewers saw another reason for seeing the infatuation as improbable--a sneaking suspicion that Niles was gay.

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Both of the Crane brothers were connoisseurs of the finer things--art, opera, wine, and gourmet food. "Before the term 'metrosexual' even entered the English language," writes critic Kamal Al-Solaylee, "Frasier and his brother typified it. The most ambiguously gay couple since Batman and Robin, Frasier and Niles's tastes veered toward the stereotypically gay while their sexual orientation remained decidedly straight"--at least according to the storyline. Al-Solaylee also notes that "the dynamic between the two brothers--protective, competitive, at times destructive--is sometimes rendered in such subtle homoerotic tones that it casts the brothers almost as lovers, or at least co-dependent partners. It's a characterization that the show's writers (many of whom are openly gay) have mined for its comic potential."

As Pierce approached his role, he "look[ed] for ways to make [his pompous character] as close to an actual human being as possible" and did so with great success. His Niles--fussy, fastidious, and fey, prone to haughtiness but vulnerable at his core--was a favorite of both the audience and the critics. Pierce was nominated for an Emmy Award as Best Supporting Actor in a Comedy Series in each of the eleven years that Frasier ran, winning four times. Other honors included awards from the Screen Actors Guild and the Television Critics Association.

During the run of Frasier, Pierce had mostly minor roles in a number of films--Andrew Bergman's Isn't She Great (2000), Pontus Löwenhielm and Patrick von Krusenstjerna's Chain of Fools (2000), David Wain's Wet Hot American Summer (2001), Steven Soderbergh's Full Frontal (2002), and Peyton Reed's Down with Love (2003). He did, however, have an important part as White House counsel John Dean in Oliver Stone's Nixon (1995).

He did voice-over work in several other movies--John Lasseter's A Bug's Life (1998), Bert Ring's The Tangerine Bear (2000), Bobby and Peter Farrelly's Osmosis Jones (2001), and Ron Clements and John Musker's Treasure Planet (2002). Pierce noted that "for voice-over, you have to energize your voice more. But I still value subtlety and throwaway delivery. That's the kind of humor that I do--not hitting anything too hard, letting people be surprised by the laugh rather than pointing the way to it."

Pierce returned to Broadway in 2004 in the musical comedy Spamalot (book and lyrics by Eric Idle, music by John DuPrez and Idle), a stage adaptation of Terry Gilliam and Terry Jones's Monty Python and the Holy Grail (1975). Pierce starred as Sir Robin, a knight fonder of minstrelsy than combat. He showed off both his singing and dancing abilities in the show-stopping production number "You Won't Succeed on Broadway," in which he and a male chorus performed a Cossack dance reminiscent of the one in Fiddler on the Roof.

Pierce next starred as Boston police lieutenant--and fan of musical theater--Frank Cioffi in Curtains (2007). The musical, originally a collaboration of composer John Kander and lyricist Fred Ebb, with a book by Peter Stone, was still unfinished when both Ebb and Stone died. Rupert Holmes took over the work on the book, largely rewriting it, and he and Kander provided additional lyrics to bring the piece to completion.

Pierce won plaudits for his portrayal of Lieutenant Cioffi, who, when called in to investigate the on-stage murder of the incompetent leading lady in a foundering musical, Robbin' Hood of the Old West, winds up not only solving the crime but also working the musical into shape and even helping various couples among the cast salvage their romantic relationships.

Reviewer David Rooney of Daily Variety wrote that "Pierce, with his polished comic timing and more than serviceable singing skills, is the most invaluable asset [to the show]. Combining a doe-eyed apparent docility with a suggestion of mischief, and balancing the seriousness of his role as investigator with his giddy distraction at being thrust into showbiz, he's clearly having a great time up there. His detective is a memorable comic creation."